PROJECT SUMMARY/ABSTRACT CADA analog TL020 has been identified as a new anti-hepatitis B lead compound in the NIAID, DMID in-vitro antiviral screening program. Its EC50 for inhibition of secreted HBV (virion) DNA was found to be 0.5-0.6 M and its EC50 for inhibition of intracellular DNA was found to be 1.2 M in the secondary screen. TL020 has relatively low cytotoxicity, with SI50 values ranging from 40-80 in these assays. CADA compounds have uncovered a novel mechanism for selectively inhibiting expression of certain proteins. The lead compound, CADA, acts as an anti-HIV agent by down-modulating CD4 on the surface of immune cells. It directly binds the signal peptide of nascent CD4 during translation and inhibits its translocation across the membrane of the endoplasmic reticulum (ER). CADA selectively binds the 25-residue signal peptide of human CD4, but not non- primate (e.g. mouse) CD4, apparently binding specific amino acid residues. It is a unique small-molecule agent that has been shown to inhibit co-translational translocation of select proteins across the ER membrane by binding its signal peptide. CADA has also been found to decrease cell-surface expression of the human protein sortilin, which is implicated in numerous diseases, including frontotemporal lobar degeneration, autism, Alzheimer's disease, atherosclerosis, and breast cancer. CADA compounds apparently bind the sortilin signal peptide with similar affinity, but the maximum efficacy is somewhat less than for CD4. After analyzing the stucture of the signal peptide of the thyroid stimulating hormone receptor (TSHR), a collaboration was initiated to test CADA compounds for decreasing expression of TSHR for treatment Graves disease. As hypothesized, compounds were identified that down-modulate TSHR, but not CD4 or sortilin! These results show the potential for tailoring the structures of compounds for selectively decreasing expression of specific proteins of therapeutic interest. CADA compounds do not inhibit any DNA polymerase, the most common mechanism of action of anti-HBV drugs. Our main hypothesis is that due to its novel mechanism of action, TL020 decreases expression of a previously unidentified host cell protein that is required for HBV replication. The Specific Aims of this proposed project address the following three overall goals: 1. Identification of the mechanism of action of TL020, including its molecular target. 2. Lead optimization by synthesis of TL020 analogs and screening anti-HBV potency in vitro. 3. Initial preclinical pharmacokinetic evaluation of potent candidates for follow-on studies in vivo. The proposed activities in this project are to use standard methods to investigate the mechanism by which TL020 inhibits HBV replication and to also examine the effects of TL020 on host protein expression. New analogs of TL020 will be synthesized to examine structure-activity relationships. These and previously synthesized CADA compounds will be screened for potency, toxicity and solubility for the purpose of lead optimization. The most selective compounds with the best physical properties will undergo pharmacokinetic evaluation for cell permeability and metabolic stability in order to identify candidates for preclinical evaluation in vivo. The proposed project fits perfectly into the high-risk high-reward model of the R21 program. The potential risks are that the mechanism of action of TL020 might not be identified or that a drug with suitable properties for in vivo studies might not be found. These are the risks of any drug development program. The potential rewards are that a new target for designing anti-HBV drugs will be identified, a practical drug for therapy of chronic HBV infection will be developed, and a new understanding of how to target signal peptides with small molecules that will enable the design of novel drugs for numerous diseases and conditions.